Mechanism Research On Apoptosis Induced By2,4-DCP In Primary Hepatocytes Of Grass Carp(Ctenopharyngodon Idella)

Chlorophenols (CPs) are widely used as wood preservatives, fungicides, pesticides and herbicides. CPs can exist stably in the environment especially in the water due to its low degradation capacity and are regarded as an important group of environmental contaminants. CPs do not only affect the growth of various aquatic organisms, they can also enter the body through the food chain and endanger human health.2,4-dichlorophenol (2,4-DCP), one of the most abundance of CPs in the environment, is used as solvents, pesticides and pharmaceutical intermediates, has been found to be harmful to humans and animals. Therefore,2,4-DCP has been defined as priority pollutants in USA, China and many other countries.2,4-DCP can affect the growth, development and reproduction of the organism, induce oxidative stress, change the expression of related genes and even lead to apoptosis.Apoptosis, also named programmed cell death, can be mediated by the mitochondrial pathway and the death receptor pathway.2,4-DCP has been demonstrated to induce apoptosis, but the detailed mechanism remains unclear. Therefore, in this study the cell viability, apoptosis, the mitochondrial membrane potential (△Ψm), the mRNA expression of apoptosis-related factors caspase-3, Bcl-2associated X protein (Bax), Bcl-2and TNF-a, the protein expression of LAP and the content of intracellular reactive oxygen species (ROS) in grass carp hepatocytes exposed to2,4-DCP were detected and measured. Exogenous mitochondrial protective agent acetyl-L-carnitine hydrochloride (ALC) was added to further explore the relationship of2,4-DCP-induced apoptosis and mitochondrial pathway. The results were as follows:1.2,4-DCP inhibited the cell viability of grass carp primary hepatocytes.3-(4,5-dimethyl-thiazol-2)-2,5-diphenyl tetrazolium bromide (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) test was used for detecting the cell viability of grass carp hepatocytes.Exposure to2,4-DCP (0.50,1.00mM) for12h significantly reduced the cell viability of grass carp hepatocytes (P<0.05).2.2,4-DCP induced apoptosis in grass carp hepatocytes. Fluorescence microscopy and flow cytometry were used to detect apoptosis of grass carp hepatocytes.Exposure ofgrass carp hepatocytes to2,4-DCP (0.50,1.00mM) for12h significantly increased the apoptosis rate (P<0.05).3.2,4-DCP decreased the△Ψm of grass carp hepatocytes. Fluorescence spectrometry was used to detect the mitochondrial membrane potential of grass carp hepatocytes. The△Ψm was significantly decreased after grass carp hepatocytes being exposed to0.10and0.50mM2,4-DCP for12h(P<0.05).4.2,4-DCP caused ROS production in grass carp hepatocytes. We detected intracellular ROS production in hepatocytes of grass carp by flow cytometry. The results showed that intracellular ROS level was much higher than that in control group when cells were treated with0.10and0.50mM2,4-DCP for5h (P<0.05). However, there were no significant differences (P>0.05) between control and2,4-DCP treatment groups (0.10and0.50mM) after exposure for12h.5.2,4-DCP changed the mRNA expression of apoptosis related genes in grass carp hepatocytes. Real-time quantitative PCR was used to detect the mRNA expression of apoptosis-related gene.0.50mM2,4-DCP increased the mRNA expression of caspase-3, TNF-a, BAX and BAX/Bcl-2mRNA ratio (P<0.05).6. ALC at5mM inhibited the2,4-DCP-induced apoptosis and significantly restored the△Ψm (P<0.05), reduced caspase-3, TNF-a and Bax mRNA expression (P<0.05) and the ratio of Bax/Bcl-2mRNA (P<0.05).7.2,4-DCPdecreased the mRNA expression of IAP, but it had no effect on IAP protein expression in grass carp hepatocytes. We designed a pair of specific primers for IAP gene by bioinformatics technology and amplified DNA fragment.Then we introduced this IAP gene fragment into E.coli to express protein which then was injected into New Zealand white rabbits to prepare antiserum. Finally, the specificanti-IAP polyclonal antibody was obtained by affinity purification. Immunoblot analysis was used to detect the IAP protein expression of grass carp hepatocytes induced by2,4-DCP. The results showed that2,4-DCP (0.10,0.50mM) had no effect on IAP protein expression in grass carp hepatocytes (P>0.05).In conclusion, the present study demonstrated that2,4-DCP-induced apoptosis in primary hepatocytes occurred through the mitochondrial pathway. This process was preceded by the generation of ROS and the disruption of△Ψm, which resulted in the increased mRNA expression of caspase-3and Bax, the elevated Bax/Bcl-2ratio, thus leading to apoptosis. The results reveals the2,4-DCP-induced apoptosis pathway and its molecular mechanism, provides scientific research basis for the research of2,4-DCP.